Antibody microarray

The concept and methodology of antibody microarrays were first introduced by Tse Wen Chang in 1983 in a scientific publication{{cite journal | author = Chang TW | title = Binding of cells to matrixes of distinct antibodies coated on solid surface | journal = J. Immunol. Methods | volume = 65 | issue = 1–2 | pages = 217–23 |date=December 1983 | pmid = 6606681 | doi = 10.1016/0022-1759(83)90318-6 }} and a series of patents,Chang, Tse W. {{US Patent|4591570}} "Matrix of antibody-coated spots for determination of antigens", Priority date February 2, 1983Chang, Tse W. {{US Patent|4829010}} "Immunoassay device enclosing matrixes of antibody spots for cell determinations", Priority date March 13, 1987Chang, Tse W. {{US Patent|5100777}} "Antibody matrix device and method for evaluating immune status", Priority date April 27, 1987 when he was working at Centocor in Malvern, Pennsylvania. Chang coined the term “antibody matrix” and discussed “array” arrangement of minute antibody spots on small glass or plastic surfaces. He demonstrated that a 10×10 (100 in total) and 20×20 (400 in total) grid of antibody spots could be placed on a 1×1 cm surface. He also estimated that if an antibody is coated at a 10 μg/mL concentration, which is optimal for most antibodies, 1 mg of antibody can make 2,000,000 dots of 0.25 mm diameter. Chang's invention focused on the employment of antibody microarrays for the detection and quantification of cells bearing certain surface antigens, such as CD antigens and HLA allotypic antigens, particulate antigens, such as viruses and bacteria, and soluble antigens. The principle of "one sample application, multiple determinations", assay configuration, and mechanics for placing absorbent dots described in the paper and patents should be generally applicable to different kinds of microarrays. When Tse Wen Chang and Nancy T. Chang were setting up Tanox, Inc. in Houston, Texas in 1986, they purchased the rights on the antibody matrix patents from Centocor as part of the technology base to build their new startup. Their first product in development was an assay, termed “immunosorbent cytometry”,{{cite journal | author = Chang TW | title = Immunosorbent Cytometry | journal = Biotechnology | volume = 11 | issue = 3 | pages = 291–3 |date=March 1993 | pmid = 7765290 | doi = 10.1038/nbt0393-291 | s2cid = 35328421 }} which could be employed to monitor the immune status, i.e., the concentrations and ratios of CD3⁺, CD4⁺, and CD8⁺ T cells, in the blood of HIV-infected individuals.

The theoretical background for protein microarray-based ligand binding assays was further developed by Roger Ekins and colleagues in the late 1980s.{{cite journal | author = Ekins RP | title = Multi-analyte immunoassay | journal = J Pharm Biomed Anal | volume = 7 | issue = 2 | pages = 155–68 | year = 1989 | pmid = 2488616 | doi = 10.1016/0731-7085(89)80079-2 }}{{cite journal |vauthors=Ekins RP, Chu FW | title = Multianalyte microspot immunoassay—microanalytical "compact disk" of the future | journal = Clin. Chem. | volume = 37 | issue = 11 | pages = 1955–67 |date=November 1991 | pmid = 1934470 | doi = 10.1016/0167-7799(94)90111-2 }}{{cite journal |author=Ekins RP |title=Ligand assays: from electrophoresis to miniaturized microarrays |journal=Clin. Chem. |volume=44 |issue=9 |pages=2015–30 |date=September 1998 |pmid=9733000 |doi= 10.1093/clinchem/44.9.2015|url=http://www.clinchem.org/cgi/pmidlookup?view=long&pmid=9733000|doi-access=free }} According to the model, antibody microarrays would not only permit simultaneous screening of an analyte panel, but would also be more sensitive and rapid than conventional screening methods. Interest in screening large protein sets only arose as a result of the achievements in genomics by DNA microarrays and the Human Genome Project.

The first array approaches attempted to miniaturize biochemical and immunobiological assays usually performed in 96-well microtiter plates. While 96-well plate-based antibody arrays have high-throughput capability, the small surface area in each well limits the number of antibody spots and thus, the number of analytes detected. Other solid supports, such as glass slides and nitrocellulose membranes, were subsequently utilized to develop arrays which could accommodate larger panels of antibodies.{{cite journal|last1=Jiang, W., Mao, Y. Q., Huang, R., Duan, C., Xi, Y., Yang, K., & Huang, R. P.|title=Protein expression profiling by antibody array analysis with use of dried blood spot samples on filter paper|journal=Journal of Immunological Methods|date=2014|volume=403|issue=1 |pages=79–86|pmid=24287424|doi=10.1016/j.jim.2013.11.016}} Nitrocellulose membrane-based arrays are flexible, easy to handle, and have increased protein binding capacity, but are less amenable to high throughput or automated processing. Chemically derivatized glass slides allow for printing of sub-microliter sized antibody spots, reducing the array surface area without sacrificing spot density. This in turn reduces the volume of sample consumed. Glass slide-based arrays, owing to their smooth and rigid structure, can also be easily fitted to high-throughput liquid handling systems.

Most antibody array systems employ 1 of 2 non-competitive methods of immunodetection: single-antibody (label-based) detection and 2-antibody (sandwich-based) detection. The latter method, in which analyte detection requires the binding of 2 distinct antibodies (a capture antibody and a reporter antibody, each binding to a unique epitope), confers greater specificity and lower background signal compared with label-based immunodetection (where only 1 capture antibody is used and detection is achieved by chemically labeling all proteins in the starting sample). Sandwich-based antibody arrays usually attain the highest specificity and sensitivity (ng – pg levels) of any array format; their reproducibility also enables quantitative analysis to be performed.{{cite journal | author = Zeng Q., Chen W. | year = 2010 | title = The functional behavior of a macrophage/fibroblast co-culture model derived from normal and diabetic mice with a marine gelatin–oxidized alginate hydrogel | journal = Biomaterials | volume = 31 | issue = 22| pages = 5772–5781 | doi=10.1016/j.biomaterials.2010.04.022| pmid = 20452666 | pmc = 2876200}}{{cite journal | author = Sohn Elliott H | display-authors = etal | year = 2015 | title = Allogenic iPSC-derived RPE cell transplants induce immune response in pigs: a pilot study | journal = Scientific Reports | volume = 5 | page = 11791 | doi=10.1038/srep11791| pmid = 26138532 | pmc = 4490339 | bibcode = 2015NatSR...511791S }} Due to the difficulty of developing matched antibody pairs that are compatible with all other antibodies in the panel, small arrays often make use of a sandwich approach. Conversely, high-density arrays are easier to develop at a lower cost using the single antibody label-based approach. In this methodology, one set of specific antibodies is used and all the proteins in a sample are labelled directly by fluorescent dyes or haptens.

Initial uses of antibody-based array systems included detecting IgGs and specific subclasses,{{cite journal | author = Silzel J. W., Cercek B., Dodson C., Tsay T., Obremski R. J. | year = 1998 | title = Mass-sensing, multianalyte microarray immunoassay with imaging detection | journal = Clin. Chem. | volume = 44 | issue = 9| pages = 2036–2043 | doi = 10.1093/clinchem/44.9.2036 | pmid = 9733002 | doi-access = free }}{{cite journal | author = Mendoza L. G., McQuary P., Mongan A., Gangadharan R., Brignac S., Eggers M. | year = 1999 | title = High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA) | journal = BioTechniques | volume = 27 | issue = 4| pages = 778–788 | doi=10.2144/99274rr01| pmid = 10524321 | doi-access = free }} analyzing antigens,{{cite journal | author = Lueking A., Horn M., Eickhoff H., Bussow K., Lehrach H., Walter G. | year = 1999 | title = Protein microarrays for gene expression and antibody screening | journal = Anal. Biochem. | volume = 270 | issue = 1| pages = 103–111 | doi=10.1006/abio.1999.4063| pmid = 10328771 }} screening recombinant antibodies,de Wildt, R. M., Mundy, C. R., Gorick, B. D., and Tomlinson, I. M. (2000) Antibody arrays for high-throughput screening of antibody-antigen interactions. Nature Biotechnol. 18, 989 –994{{cite journal | author = Holt L. J., Bussow K., Walter G., Tomlinson I. M. | year = 2000 | title = By-passing selection: Direct screening for antibody–antigen interactions using protein arrays | journal = Nucleic Acids Res | volume = 28 | issue = 15| page = E72 | doi=10.1093/nar/28.15.e72| pmc = 102691 | pmid=10908365}} studying yeast protein kinases,{{cite journal | author = Zhu H., Klemic J. F., Chang S., Bertone P., Casamayor A., Klemic K. G., Smith D., Gerstein M., Reed M. A., Snyder M. | year = 2000 | title = Analysis of yeast protein kinases using protein chips | journal = Nature Genetics | volume = 26 | issue = 3| pages = 283–289 | doi=10.1038/81576| pmid = 11062466 | s2cid = 9238048 }} analyzing autoimmune antibodies,{{cite journal | author = Joos T. O., Schrenk M., Hopfl P., Kroger K., Chowdhury U., Stoll D., Schorner D., Durr M., Herick K., Rupp S., Sohn K., Hammerle H. | year = 2000 | title = A microarray enzyme-linked immunosorbent assay for autoimmune diagnostics | journal = Electrophoresis | volume = 21 | issue = 13| pages = 2641–2650 | doi=10.1002/1522-2683(20000701)21:13<2641::aid-elps2641>3.0.co;2-5| pmid = 10949141 | s2cid = 24008668 }} and examining protein-protein interactions.{{cite journal | author = Walter G., Bussow K., Cahill D., Lueking A., Lehrach H. | year = 2000 | title = Protein arrays for gene expression and molecular interaction screening | journal = Curr. Opin. Microbiol. | volume = 3 | issue = 3| pages = 298–302 | doi=10.1016/s1369-5274(00)00093-x| pmid = 10851162 }}{{cite journal | author = Service R. F. | year = 2000 | title = Biochemistry: Protein arrays step out of DNA's shadow | journal = Science | volume = 289 | issue = 5485| page = 1673 | doi=10.1126/science.289.5485.1673| pmid = 11001728 | s2cid = 2753950 }}{{cite journal | author = Wang Y., Wu T. R., Cai S., Welte T., Chin Y. E. | year = 2000 | title = Stat1 as a Component of Tumor Necrosis Factor Alpha Receptor 1-TRADD Signaling Complex To Inhibit NF-κB Activation | journal = Molecular and Cellular Biology | volume = 20 | issue = 13| pages = 4505–4512 | doi=10.1128/mcb.20.13.4505-4512.2000| pmid = 10848577 | pmc = 85828 }} The first approach to simultaneously detect multiple cytokines from physiological samples using antibody array technology was by Ruo-Pan Huang and colleagues in 2001.R.-P. Huang. (2001). Simultaneous detection of multiple proteins with an array-based enzyme-linked immunosorbant assay (ELISA) and enhanced chemiluminescence (ECL). Clin. Chem. Lab. Med. 39:209-214. Their approach used Hybond ECL membranes to detect a small panel of 24 cytokines from cell culture conditioned media and patient's sera and was able to profile cytokine expression at physiological levels. Huang took this technology and started a new business, RayBiotech, Inc., the first to successfully commercialize a planar antibody array.

In the last ten years, the sensitivity of the method was improved by an optimization of the surface chemistry as well as dedicated protocols for their chemical labeling.{{cite journal |vauthors=Kusnezow W, Banzon V, Schröder C, Schaal R, Hoheisel JD, Rüffer S, Luft P, Duschl A, Syagailo YV| year = 2007 | title = Antibody microarray-based profiling of complex specimens: systematic evaluation of labeling strategies | journal = Proteomics | volume = 7 | issue = 11| pages = 1786–99 | doi=10.1002/pmic.200600762 | pmid=17474144| s2cid = 9852887 }} Currently, the sensitivity of antibody arrays is comparable to that of ELISA{{cite journal |vauthors=Kusnezow W, Banzon V, Schröder C, Schaal R, Hoheisel JD, Rüffer S, Luft P, Duschl A, Syagailo YV| year = 2007 | title = Antibody microarray-based profiling of complex specimens: systematic evaluation of labeling strategies | journal = Proteomics | volume = 7 | issue = 11| pages = 1786–99 | doi=10.1002/pmic.200600762 | pmid=17474144| s2cid = 9852887 }}{{cite journal | author = Wingren Christer, Ingvarsson Johan, Dexlin Linda, Szul Dominika, Borrebaeck Carl A. K. | year = 2007 | title = Design of recombinant antibody microarrays for complex proteome analysis: Choice of sample labeling-tag and solid support | journal = Proteomics | volume = 7 | issue = 17| pages = 3055–3065 | doi=10.1002/pmic.200700025 | pmid=17787036| s2cid = 29548647 }} and antibody arrays are regularly used for profiling experiments on tissue samples, plasma or serum samples and many other sample types. One main focus in antibody array based profiling studies is biomarker discovery, specifically for cancer.Alhamdani, MS; Schröder, C; Hoheisel, JD (Jul 6, 2009). "Oncoproteomic profiling with antibody microarrays". Genome medicine 1 (7): 68{{cite journal | author = Jones V. S., Huang R. Y., Chen L. P., Chen Z. S., Fu L., Huang R. P. | year = 2016 | title = Cytokines in cancer drug resistance: cues to new therapeutic strategies | journal = Biochimica et Biophysica Acta (BBA) - Reviews on Cancer | doi = 10.1016/j.bbcan.2016.03.005 | pmid = 26993403 | volume = 1865 | issue = 2| pages = 255–265 | doi-access = free }}{{cite journal | author = Burkholder B., Burgess R.Y. R., Luo S.H., Jones V.S., Zhang W.J., Lv Z.Q., Gao C.-Y., Wang B.-L., Zhang Y.-M., Huang R.-P. | year = 2014 | title = Tumor-induced perturbations of cytokines and immune cell networks | journal = Biochimica et Biophysica Acta (BBA) - Reviews on Cancer | doi = 10.1016/j.bbcan.2014.01.004 | pmid = 24440852 | volume = 1845 | issue = 2| pages = 182–201 | doi-access = free }}{{cite journal | author = Lin Y. |author2=Luo S. |author3=Shao N. |author4=Wang S. |author5=Duan C. |author6=Burkholder B.|display-authors=et al | year = 2013 | title = Peeking into the Black Box: How Cytokine Antibody Arrays Shed Light on Molecular Mechanisms of Breast Cancer Development and its Treatment | journal = Current Proteomics | volume = 10 | issue = 4| pages = 269–277 | doi=10.2174/1570164610666131210233343}}{{cite journal | author = Huang R.-P. | year = 2007 | title = An array of possibilities in cancer research using cytokine antibody arrays | journal = Expert Review of Proteomics| volume = 4 | issue = 2| pages = 299–308 | doi=10.1586/14789450.4.2.299| pmid = 17425464 | s2cid = 30102746 }} For cancer-related research, the development and application of an antibody array comprising 810 different cancer-related antibodies was reported in 2010.{{cite journal |vauthors=Schröder C, Jacob A, Tonack S, Radon TP, Sill M, Zucknick M, Rüffer S, Costello E, Neoptolemos JP, Crnogorac-Jurcevic T, Bauer A, Fellenberg K, Hoheisel JD| year = 2010 | title = Dual-color proteomic profiling of complex samples with a microarray of 810 cancer-related antibodies | journal = Molecular & Cellular Proteomics | volume = 9 | issue = 6| pages = 1271–80 | doi=10.1074/mcp.m900419-mcp200| doi-access = free | pmid = 20164060 | pmc = 2877986}} Also in 2010, an antibody array comprising 507 cytokines, chemokines, adipokines, growth factors, angiogenic factors, proteases, soluble receptors, soluble adhesion molecules, and other proteins was used to screen the serum of ovarian cancer patients and healthy individuals and found a significant difference in protein expression between normal and cancer samples.{{cite journal | author = Huang R., Jiang W., Yang J., Mao Y.Q., Zhang Y., Yang W., Yang D., Burkholder B., Huang R.F., Huang R.P. | year = 2010 | title = A biotin label-based antibody array for high-content profiling of protein expression | journal = Cancer Genomics Proteomics | volume = 7 | issue = 3| pages = 129–41 | pmid = 20551245 }} More recently, antibody arrays have helped determine specific allergy-related serum proteins whose levels are associated with glioma and can reduce the risk years before diagnosis.{{cite journal | author = Schwartzbaum J. |author2=Seweryn M. |author3=Holloman C. |author4=Harris R. |author5=Handelman S. K. |author6=Rempala G. A.|display-authors=et al | year = 2015 | title = Association between Prediagnostic Allergy-Related Serum Cytokines and Glioma | journal = PLOS ONE | volume = 10 | issue = 9| page = e0137503 | doi=10.1371/journal.pone.0137503|pmid=26352148 | pmc=4564184 |bibcode=2015PLoSO..1037503S |doi-access=free }} Protein profiling with antibody arrays have also proven successful in areas other than cancer research, specifically in neurological diseases such as Alzheimer's. A number of studies have attempted to identify biomarker panels that can distinguish Alzheimer's patients, and many have used antibody arrays in this process. Jaeger and colleagues measured nearly 600 circulatory proteins to discover biological pathways and networks affected in Alzheimer's and explored the positive and negative relationships of the levels of those individual proteins and networks with the cognitive performance of Alzheimer's patients.{{cite journal | author = Jaeger P. A. |author2=Lucin K. M. |author3=Britschgi M. |author4=Vardarajan B. |author5=Huang R.-P. |author6=Kirby E. D.|display-authors=et al | year = 2016 | title = Network-driven plasma proteomics expose molecular changes in the Alzheimer's brain | journal = Molecular Neurodegeneration | volume = 11 | issue = 1| page = 31 | doi=10.1186/s13024-016-0105-4| pmc = 4877764 | pmid=27216421 |doi-access=free }} Currently the largest commercially available sandwich-based antibody array detects 1000 different proteins.RayBiotech, Inc. Antibody Arrays. (2017). Retrieved from the RayBiotech, Inc. website http://www.raybiotech.com/antibody-array.html In addition, antibody microarray based protein profiling services are available analyzing protein abundance and protein phosphorylation or ubiquitinylation status of 1030 proteins in parallel.{{Cite web|url=http://www.sciomics.de/services/protein-phosphorylation-analysis|title=Sciomics: Antibody meets microarray - scioPhospho|website=www.sciomics.de|language=en-us|access-date=2018-04-24}}

Antibody arrays are often used for detecting protein expression from many sample types, but also in those with various preparations. Jiang and colleagues illustrated nicely the correlation between array protein expression in two different blood preparations: serum and dried blood spots.{{cite journal|last1=Jiang, W., Mao, Y. Q., Huang, R., Duan, C., Xi, Y., Yang, K., & Huang, R. P.|title=Protein expression profiling by antibody array analysis with use of dried blood spot samples on filter paper|journal=Journal of Immunological Methods|date=2014|volume=403|issue=1 |pages=79–86|pmid=24287424|doi=10.1016/j.jim.2013.11.016}} These different blood sample preparations were analyzed using three antibody array platforms: sandwich-based, quantitative, and label-based, and a strong correlation in protein expression was found, suggesting that dried blood spots, which are a more convenient, safe, and inexpensive means of obtaining blood especially in non-hospitalized public health areas, can be used effectively with antibody array analysis for biomarker discovery, protein profiling, and disease screening, diagnosis, and treatment.

Using antibody microarray in different medical diagnostic areas has attracted researchers attention. Digital bioassay is an example of such research domains. In this technology, an array of microwells on a glass/polymer chip are seeded with magnetic beads (coated with fluorescent tagged antibodies), subjected to targeted antigens and then characterised by a microscope through counting fluorescing wells. A cost-effective fabrication platform (using OSTE polymers) for such microwell arrays has been recently demonstrated and the bio-assay model system has been successfully characterised.{{cite journal|doi=10.1021/acsami.6b15415 | volume=9 | issue=12 | title=Single-Step Imprinting of Femtoliter Microwell Arrays Allows Digital Bioassays with Attomolar Limit of Detection | year=2017 | journal=ACS Applied Materials & Interfaces | pages=10418–10426 | author=Decrop Deborah | pmid=28266828| url=https://lirias.kuleuven.be/bitstream/123456789/576695/1/ACSAppMatInt_DecropD_Imprinting%20femtoliter%20microwell_revised3.docx | url-access=subscription }} Furthermore, immunoassays on thiol-ene "synthetic paper" micropillar scaffolds have shown to generate a superior fluorescence signal.{{cite journal | doi=10.1016/j.bios.2020.112279 | title=Immunoassays on thiol-ene synthetic paper generate a superior fluorescence signal |year=2020 |journal=Biosensors and Bioelectronics | last1 = Guo | first1 = W | last2 = Vilaplana | first2 = L | last3 = Hansson | first3 = J | last4 = Marco | first4 = P| last5 = van der Wijngaart | first5 = W | volume=163 | page=112279 | pmid=32421629 | s2cid=218688183 | hdl = 10261/211201 | hdl-access = free }}

• ELISA
• Protein microarray
• DNA microarray
• Tissue microarray
• Chemical compound microarray
• Microarray imprinting and surface energy patterning

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